Fig 1: Upregulation of XBP1s promotes SKI PASMC proliferation. (A) Xbp1 mRNA in PASMCs. n = 5–9. (B) Representative images of XBP1s immunofluorescence staining (red) in PASMCs and quantitative analysis of fluorescence density in graph. Nuclei are indicated by DAPI (blue). n = 5. Scale bar: 50 µm. (C) Representative images of XBP1s immunofluorescence staining (green) in pulmonary vessels indicated by a-SMA staining (red), and quantitative analysis of fluorescence density in graph. n = 5. The white arrow indicates that XBP1s is mainly expressed in the remodeled pulmonary vessels. Scale bar: 50 µm. (A)?(C), *P < 0.05, SKI vs. WT, unpaired t-test. (D) Representative Western blot of WT PASMCs overexpressed Xbp1s plasmid or vector control (Ctrl), and quantification of band intensities in graph. *P < 0.05, Xbp1s vs. Ctrl, unpaired t-test, n = 3–7. (E) The relative percentage of XBP1s (red) or Ki67-positive (green) cells in WT PASMCs overexpressed Xbp1s plasmid or vector control (Ctrl). *P < 0.05, Xbp1s vs. Ctrl, unpaired t-test, n = 5. Scale bar: 100 µm. (F) Cell proliferation in WT PASMCs. *P < 0.05, Xbp1s vs. Ctrl, unpaired t-test, n = 5. Data were presented as mean ± SEM.
Fig 2: Knockdown of TM9SF4 promoted inflammation, increased ER stress, and increased cell deaths in colon epithelial cells. (A) Expression levels of TM9SF4 in HCECs, Caco-2, and HT-29 cells were compared by Western blot (n = 3). HCECs with Scr-shRNA or TM9SF4-shRNAs were challenged with (B) 100 ng/mL LPS or (C) 1 µg/mL PGN for 24 hours. The mRNA levels of TNFa, IL1ß, and IL6 were assessed by qRT-PCR (n = 3). (D) HCECs with Scr-shRNA or TM9SF4-shRNAs were challenged with LPS. The levels of TM9SF4, GRP78, ATF4, cleaved ATF6, spliced XBP1, CHOP, COX2, and cleaved caspase 3 were detected by Western blot. Representative from 3 experiments. (E) ROS measurement in HCECs by DHE fluorescence. Representative from 4 experiments. Scale bar: 100 µm. (F) Representative FACS analysis and data summary (right) of Annexin V and PI staining in LPS-challenged HCECs (n = 4). KEGG analysis of altered signaling pathways after TM9SF4 knockdown in (G) basal condition (vehicle) and (H) inflammatory condition (LPS) in HCECs. HCECs were transfected with scrambled-shRNA (SCR) or TM9SF4-shRNA 1 (KD) for RNA sequencing analysis. Shown are means ± SEM. *P < .05, **P < .01, and ***P < .001. ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRH, gonadotropin releasing hormone; KD, knockdown clone; PI, propidium iodide; SCR, scrambled control; TGF, transforming growth factor; TRP, trasient receptor potential.
Fig 3: Inhibition of IRE1a/XBP1s pathway prevents SKI pulmonary vascular remodeling. (A) Summary of the severity of vascular remodeling in PAs and veins. (B) The protein expression of XBP1 and cell cycle related proteins in lungs. *P < 0.05, SKI treated with solvent control (SKI/Ctrl) vs. WT treated with solvent control (WT/Ctrl), #P < 0.05, SKI treated with 4µ8C (SKI/4µ8C) vs. SKI/Ctrl. ANOVA with Bonferroni correction, n = 9. The WT/Ctrl group (n = 9) includes the data from Fig. S8 (n = 5). Data were presented as mean ± SEM.
Fig 4: ASFV K205R induces ER stress. (A,B) 3D4/21 (A) and HeLa (B) cells were transfected with K205R-HA plasmid as indicated for 24 h. Bip, p-PERK, PERK, p-eIF2a, eIF2a, ATF4, ATF6, XBP1, K205R-HA, and ß-actin were assessed with immunoblotting analysis. (C–G) 3D4/21 cells were transfected with K205R-HA plasmid as indicated for 24 h. The mRNA levels of ERdj4 (C), Xbp1(s)/Xbp1(t) (D), Atf4 (E), Gadd34 (F), and Chop (G) were assessed with qRT-PCR analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. (H) 3D4/21 cells were transfected with K205R-HA plasmid and treated with GSK2606414 (GSK, 10 µM) as indicated for 24 h. p-PERK, PERK, p-eIF2a, eIF2a, K205R-HA, and ß-actin were assessed with immunoblotting analysis.
Fig 5: KO of TM9SF4 promoted M1 macrophage polarization but inhibited M2 macrophage polarization. (A and B) Colonic CD11b+ cells from 2% DSS-challenged WT or KO mice were purified by magnetic-activated cell sorting. The mRNA levels of (A) M1 macrophages markers IL23p19, IL6, iNOS, and (B) M2 macrophages markers MRC1, Ym1, Arg1 were assessed by qRT-PCR (n = 4). Representative images and data summary for immunohistochemical staining of (C) CD80 and (D) CD206 in the colon tissues of DSS-treated WT/KO mice (n = 7). Brown, CD80/CD206-positive signals; blue, nuclear counterstain. Scale bars: 100 µm. (E) Expression levels of TM9SF4 in BMDMs and Raw264.7 macrophages were compared by Western blot (n = 3). BMDMs were treated with LPS/IFN? or vehicle, followed by (F) qRT-PCR–based mRNA level analysis of IL23p19, IL6, IL12a, and iNOS (n = 4–6) or by (H) FACS analysis of CD11c+ M1 BMDMs (n = 4). (G and I) BMDMs were treated with IL4/IL13 or vehicle, followed by (G) qRT-PCR–based mRNA level analysis of Mrc1, Ym1, Il10, and Arg1 (n = 4–6) or by (I) FACS analysis of CD206+ M2 BMDMs (n = 4). (J) BMDMs were challenged with LPS/IFN?. The levels of GRP78, ATF4, cleaved ATF6, spliced XBP1, CHOP, IL1ß, and iNOS were detected by Western blot (n = 3). (K) Enhanced aggresome formation in BMDMs from KO mice by flow cytometry analysis (n = 5). Means ± SEM. *P < .05, **P < .01, and ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MFI, mean fluorescent intensity.
Supplier Page from Proteintech Group Inc for XBP-1U specific antibody